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Abstract

Background

This study investigated the influence of 2-months ingestion of an "immune" nutrient
fortified breakfast cereal on immune function and upper respiratory tract infection
(URTI) in healthy children during the winter season.

Methods

Subjects included 73 children (N = 42 males, N = 31 females) ranging in age from 7
to 13 years (mean ± SD age, 9.9 ± 1.7 years), and 65 completed all phases of the study.
Subjects were randomized to one of three groups--low, moderate, or high fortification--with
breakfast cereals administered in double blinded fashion. The "medium" fortified cereal
contained B-complex vitamins, vitamins A and C, iron, zinc, and calcium, with the
addition of vitamin E and higher amounts of vitamins A and C, and zinc in the "high"
group. Immune measures included delayed-typed hypersensitivity, global IgG antibody
response over four weeks to pneumococcal vaccination, salivary IgA concentration,
natural killer cell activity, and granulocyte phagocytosis and oxidative burst activity.
Subjects under parental supervision filled in a daily log using URTI symptoms codes.

Results

Subjects ingested 3337 ± 851 g cereal during the 2-month study, which represented
14% of total diet energy intake and 20-85% of selected vitamins and minerals. Despite
significant increases in nutrient intake, URTI rates and pre- to- post-study changes
in all immune function measures did not differ between groups.

Conclusions

Data from this study indicate that ingestion of breakfast cereal fortified with a
micronutrient blend for two winter months by healthy, growing children does not significantly
influence biomarkers for immune function or URTI rates.

Background

Nutrients are involved in the immune response to pathogens, facilitating cell division
and the production of specific antibodies and cytokines, and in providing metabolic
support for skin and mucosa physical barriers [1]. Enzymes in immune cells require the presence of micronutrients, and critical roles
have been defined for nearly all nutrients, including zinc, iron, copper, selenium,
and vitamins A, C, E, D, and B6 [2,3]. One of the earliest interactions between nutrition, immunity, and infection was
established in malnourished children [4,5]. Nutritional deficiencies and immune dysfunction, with improvements measured following
immunonutrition support, have been linked in several other groups including the frail
elderly, patients experiencing surgery, illness, and trauma, and human immunodeficiency
virus (HIV) infected individuals [6-13].

Less clear is the immune-related benefit of nutrient supplementation among healthy
children and adults with no overt signs of immune system deficiencies. Among free-living
adults, a wide variance in self-selected diet intake is compatible with normal immune
function [14-16]. High compared to low self-selected intake of vitamins through diet or supplements
by adults is not associated with altered risk of pneumonia [17,18]. Although data are limited, mixed or single micronutrient supplementation among healthy,
community-dwelling adults is largely ineffective in altering innate or adaptive immune
function, or in lowering respiratory infection rates [19-24].

Zinc, iron, and/or vitamin A, C, and E supplementation in young, malnourished or diseased
children in developing countries reduces respiratory infection morbidity and helps
counter impaired immunity [25-31]. The influence of mixed micronutrient supplementation on immune function and incidence
of upper respiratory tract infections (URTI) in healthy children is largely unstudied
[32]. Children suffer from a high rate of URTI, and the physiologic stress of rapid growth
and suboptimal dietary quality may provide room for immune benefit through micronutrient
supplementation [3]. We hypothesized that school-aged children would experience improvements in innate
and adaptive immune function and a reduction in URTI during two winter months of supplementation
with a mixture of immune-related micronutrients administered through a fortified breakfast
cereal.

Methods

Subjects and research design

Seventy-three children (42 boys and 31 girls) ranging in age from 7 to 13 years, and
in body mass index from 13 to 36 kg/m2, were recruited from local elementary schools and home school programs. Inducements
included subject stipends and free results of fitness, body composition, and immune
function tests. This study was conducted according to the guidelines laid down in
the Declaration of Helsinki and all procedures involving human subjects/patients were
approved by the university's Institutional Review Board for Human Studies. Written
informed consent was obtained from all subjects and a parent. A parent for each child
attended all orientation and test sessions, and assumed responsibility for home feeding,
dietary recording, health logs, and transportation of their children to the laboratory.

Triceps and subscapular skinfolds were measured in each child and summed using the
procedures of Lohman et al. [33]. The skinfolds were measured by one trained technician using a Lange skinfold caliper
(Cambridge Scientific Industries, Cambridge, MA).

Subjects were tested for immune function pre-study, and then again two months later
following a regimen of micronutrient supplementation through ingestion of a fortified
breakfast cereal. The subjects used a daily health log to record symptoms of sickness
using number codes. A pneumococcal vaccine was administered halfway through the study,
with the antibody response measured one month later (post-study).

Subjects were given two coded boxes for each week of the study, with instructions
to consume two to three measured cups (~50-70 g) per day (anytime of the day) for
two months. Intake was recorded in daily logs by the subjects/parents, and all unconsumed
cereal was returned to investigators for weighing to determine actual intake. To remain
in the study, subjects had to consume a minimum of 1,700 g cereal during the study
period. Subjects (with parental supervision) were instructed to avoid all other forms
of nutrient supplements during the 2-month study.

Diet intake was estimated through 3-day food records pre-study, and then again after
one and two months. The study dietitian provided detailed instructions using food
models and volume measuring supplies to subjects and parents regarding methods for
recording volume and portion size in the 3-day food records, and then entered the
information into a computerized dietary analysis system, Food Processor Plus (ESHA
Research, Salem, Oregon).

Blood and saliva sample collection

Children and parents reported to the testing facility between the hours of 7:30-9:30
am having avoided energy intake for the previous 9 h. A 4-min timed saliva sample
was first collected followed by a blood sample drawn by trained pediatric phlebotomists.
A delayed-type hypersensitivity (DTH) skin test was administered using the Mantoux
method with three antigens (Candida albicans, mumps antigen, and tetanus toxoid). Each subject reported back to the testing lab
two days later for a 48-h measure of skin induration. One month later, subjects returned
to the testing lab, and were given a pneumococcal vaccination by medical personnel.
At the end of the two-month period, saliva and blood samples were again collected,
and the DTH skin test readministered.

Immune assay measurements

All blood samples were obtained from an antecubital vein from the children while in
the supine position after having rested for more than 15 min. Routine complete blood
counts were performed by a clinical hematology laboratory (Lab Corp, Burlington, NC),
and provided leukocyte subset counts.

Lymphocyte subsets

The proportions of T cells (CD3+), B cells (CD19+), and NK cells (CD3-CD16+CD56+) were determined in whole blood preparations and absolute numbers calculated using
CBC data to allow group comparisons on blood concentrations of cells. Lymphocyte phenotyping
was accomplished by two-color fluorescent labeling of cell surface antigens with mouse
anti-human monoclonal antibodies conjugated to fluoresceinisothiocyanate (FITC) and
phycoerythrin (PE) using Simultest monoclonal antibodies and isotype controls (Becton
Dickinson, San Jose, CA). For immunophenotyping, 50 μl aliquots of heparinized whole
blood from each sample were added to five wells of a 96 well plate. Five μg (diluted
in 50 μl RPMI ) of each antibody or isotype control were added to appropriate wells
for 20 min on ice in the dark with orbital shaking (170 rpm). The cell suspension
was then lysed using 200 μl FACSlyse solution (Becton Dickinson) for 10 min in the
dark on ice with shaking. Plates were then centrifuged for five min (Beckman GS-R6
Centrifuge) at 1500 × g. Samples were kept at 4°C in the dark until analyzed by flow
cytometry (FacsCalibur, Becton Dickinson, San Jose, CA)

Natural killer cell activity (NKCA)

NKCA was assessed using the chromium release assay [34,35]. Peripheral blood mononuclear cells were isolated from heparinized blood by density
gradient centrifugation with Ficoll and sodium diatrivoate (American Red Cross, Washington
D.C.). 51Chromium labeled K562 target cells were then added (1 × 104) to each of the wells containing effector cells to yield 40:1 and 20:1 effector to
target (E:T) ratios. The assay was performed in triplicate in "V" bottom microtiter
plates (Costar, Cambridge, MA). The microtiter plates were then incubated for 4 h
at 37°C in a 5% C02 (in air) incubator. At the end of the incubation, the plates were
centrifuged for 5 min at 1500 rpm, the supernatants harvested onto Skatron harvesting
frames (Skatron, Sterling, VA), and the level of radioactivity measured in a Packard
Tri Carb Liquid Scintillation Analyzer model 2500 TR series (Packard Instruments Company,
Meriden, CT). Total release of 51Cr was determined by counting an equal aliquot of resuspended cells in 100 μl of 1%
triton-X (Sigma, St. Louis, MO). Spontaneous release was determined by counting the
radioactivity in the supernatant of labeled target cells cultured in medium alone.
The percent lysis was calculated using the mean counts per min (cpm) of triplicate
values for each E:T ratio and the following formula:

Granulocyte phagocytosis and oxidative burst activity

The phagocytosis assay utilized a FITC-labeled bacteria (Staphylococcus aureus; Molecular Probes, Eugene, OR) to quantify the degree of phagocytosis by granulocytes,
as described in a previous publication [34]. Briefly, to determine the extent of oxidative burst exhibited by granulocytes, we
employed 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Molecular Probes), a
non-fluorescent molecule which is oxidized to green fluorescent dichlorofluorescein
(DCF) as oxygen radicals are generated in the oxidative burst to kill unlabeled Staphylococcus aureus. The white blood cell count was acquired using the Becton Dickinson Unopet manual
counting protocol. Using two-color flow cytometric immunophenotying (CD45-FITC/CD13,14-PE),
the granulocyte percentage was determined. Bioparticle reagents of unlabeled and labeled
Staphylococcus aureus were suspended into PBS at a working concentration of 3 × 105 bioparticles/μl. After determining the number of phagocytic cells in 100 μl of whole
blood, and adding 15 FITC-labeled bacteria per cell, the mean channel fluorescence
(FITC) was analyzed to determine the degree of engulfed bacteria (non-phagocytized
bacteria were quenched with ethidium bromide; final concentration of 200 μM). To determine
the oxidative burst activities, either DCF-DA (final concentration, 100 μM) (basal
activity level), or DCF-DA and unlabeled bacteria (stimulated activity level) were
added to 100 μl whole blood. After incubating the samples for 60 min (37°C) in the
dark, lysing the RBC, centrifuging, and resuspending the pellets, the samples (10,000
phagocytes) were acquired on the flow cytometer.

Salivary IgA

Unstimulated saliva was collected for four min into 5 mL plastic, sterilized vials.
Participants were urged to pass as much saliva as possible into the vials during the
4-min timed session. Saliva volume was measured to the nearest 0.1 mL, and then frozen
at -80°C until analysis. Salivary IgA was measured by enzyme linked immunosorbent
assay (34). The data were expressed as concentration of sIgA (μg . mL-1), concentration of sIgA relative to total protein concentration (μg . mg-1), and salivary immunoglobulin secretion rate (μg . min-1).

Delayed-type hypersensitivity (DTH) skin response

The DTH skin response was assessed with use of three antigens, Candida albicans, mumps antigen, and tetanus toxoid (diluted 5:1), by the Mantoux method with needle
and syringe (Allermed Laboratories, Inc., San Diego, CA; Aventis Pasteur, Swiftwater,
PA). The volar surfaces of the left and right arms were cleansed and labeled (mumps
antigen and tetanus toxoid on the left arm, Candida albicans on the right). At each
site, a needle was inserted into the skin at a 45 degree angle to a depth of <0.2
mm, and 0.1 mL of antigen injected until a 5 mm pea-sized bleb was produced. After
48 h, subjects returned to the lab, and the DTH response at each test site measured.
The extent of the induration response was palpated at the reaction area (manifested
as firmness and redness), outlined with a black-ink ballpoint pen, and then removed
with scotch tape prior to mounting on the skin test record form. The tape impress
for each induration was measured across two diameters and averaged.

Pneumoccocal vaccination and IgG antibody response

Plasma samples were collected one month before and one month after the children were
administered a single 0.5 mL dose of PNEUMOVAX 23 intramuscularly (deltoid muscle)
(Merck & Co., Inc., West Point, PA). The plasma samples were assayed for specific
IgG antibodies against Pneumococcal Capsular Polysaccharide (PCP) (BINDAZYME™ Anti-PCP
IgG Enzyme Immunoassay Kit, MK012, The Binding Site LTD, Birmingham, England). The
measuring range of the assay for anti-PCP IgG antibodies levels is 3.3-270 mg/l, with
an intra-assay precision of 3.1-5.9% CV and an analytical sensitivity of 0.62 mg/l.

Upper respiratory tract infection log

Subjects with parental assistance recorded URTI symptoms on a daily basis in a log
using numbered codes. The following health problems were recorded, in accordance with
previous investigations by our research team [36]: 1) No health problems; 2) Cold symptoms (runny, stuffy nose, sore throat, coughing,
sneezing, colored discharge); 3) Flu symptoms (fever, headache, general aches and
pains, fatigue and weakness, chest discomfort, cough); 4) Nausea, vomiting, and/or
diarrhea; 5. Muscle, joint, or bone problems/injury; 6) Other health problems. An
URTI episode was recorded if cold (#2 item) or flu (#3) symptoms persisted for two
days or longer. The primary outcome reported in this study is the total days with
URTI symptoms. URTI severity and duration per episode were not monitored or calculated
in this study.

Statistical methods

Data are reported as mean ± SD, and were analyzed using SPSS 11.5 (SPSS Inc., Chicago,
IL). The dietary intake data were analyzed using 3 (groups) × 3 (times of measurement)
repeated measures ANOVA, with immune data analyzed using a 3 × 2 repeated measures
MANOVA. When Box's M suggested a violation of homoscedasticity assumption in MANOVA,
Pillais trace statistic was used as the test statistic because it has been shown to
be robust against departures from covariance equality. If the group × time interaction
P value was ≤ 0.05, a change variable was calculated and compared between groups using
Bonferroni adjusted Student's t-tests. URTI data from the daily logs were combined
into group averages and the total number of sick days recorded was compared between
groups using oneway ANOVA. Chi-square analysis was used to compare the number of children
across groups who reported at least one URTI episode during the study period. Power
analysis was performed after the study was conducted using the fpower macro in SAS
(SAS Institute, Inc., Cary, NC), and revealed that 41 to 142 subjects would be needed
(depending on the immune measure) to achieve 80% power. The power analysis is a conservative
estimate and was conducted a posteriori because data on children for all of the immune measures used in this study were not
available prior to the study. We acknowledge that the sample size is considered marginal
for this study, but subjects were well randomized, and the results are in accordance
with what we and other have observed in adults.

Results

Seventy-three children (55% male, 45% female) started the study, and 65 adhered to
all aspects of the study design and were included in the statistical analysis. Groups
did not differ significantly in subject characteristics, and data are provided in
Table 1 for all 65 subjects completing the study. Subjects consumed an average of 3337 ±
851 g cereal during the two month study, and this quantity did not vary significantly
between groups. Nutrient intake (combining self-selected diet and fortified cereal)
is summarized in Table 2 for each group. In comparison to the low group, intake of B-complex vitamins, vitamin
A, zinc, and iron was elevated in the medium and high groups at 1- and 2-months, with
vitamins E and C elevated in the high group. Figure 1 depicts the percentage contribution of the fortified cereal to total nutrient intake.
The cereal supplement represented 13.4 ± 6.6, 15.3 ± 4.3, and 12.7 ± 2.9% of total
energy intake during the study period for low, medium, and high groups, respectively,
and contributed ~20% to 85% of the nutrients listed.

Table 2. Nutrient intake data (mean ± SD, from 3-day food records taken pre-study, and after
one-month and two-months ingestion of cereal product (low-, medium, and high fortification)
in children

Figure 1.The percentage contribution of the fortified cereal to total nutrient intake after
one and two months supplementation.

The mean total number of days with URTI during the study period did not differ significantly
between groups, and subjects averaged 8.8 ± 10.9, 10.4 ± 6.5, and 7.4 ± 7.2 days with
URTI for low, medium, and high groups, respectively (p = 0.556). The percentage of
children reporting at least one URTI episode during the study period did not differ
between groups: 83%, 90%, and 74% for low, medium, and high groups, respectively (χ2 = 1.78, P = 0.411).

Discussion

Despite significant improvements in dietary intake of immune-related nutrients through
ingestion of a fortified cereal (~60 g/day) during a 2-month winter period, URTI rates
and pre- to- post-study changes in all immune function measures did not differ between
groups. These data do not support the use of micronutrient fortified breakfast cereals
by healthy, growing children for the purpose of augmenting innate or adaptive immune
function and lowering URTI risk.

Although dietary nutrients are important for immunocompetence in all humans [1], related benefits from micronutrient supplements are most often reported in populations
with immune dysreglation such as malnourished children, the frail elderly, diseased
individuals, and surgical or trauma patients [5-13]. Among healthy adults, immune function remains robust over a wide range of diet quality
and nutrient intake, and adding supplemental nutrients beyond what is obtained in
the traditional food supply has little if any discernable influence on immunity and
infection rates [14-22]. In a double-blinded, randomized, placebo-controlled study of 138 healthy adults
aged 40-80 years, Wolvers et al. [21] reported no effect of 10-weeks supplementation with a micronutrient mix (vitamins
E and C, β-carotene, and zinc) on a range of immunological measures including the
antibody response to vaccination, phagocytosis and oxidative burst activity, and lymphocyte
proliferation. An enhanced DTH response was reported, but this benefit was limited
to the older subjects, similar to data previously reported by Bogden et al. [37]. Other studies of healthy adults with uncompromised immune systems using single-nutrient
supplements such as zinc, selenium, or vitamin E have also reported negligible immune
benefits, although tocotrienol supplementation may improve the immune response to
a vaccine challenge [19-24,38].

To our knowledge, our study is the first to investigate the influence of micronutrient
supplementation through breakfast cereal fortification on immune function and URTI
in healthy, school-aged youth. Kutukculer et al. [32] studied the effect of acute, large dose vitamin A and E supplementation on the IgG
response to tetanus toxoid immunization in healthy infants, and reported no supplementation
effect on the antibody response. Several other studies have reported positive immunological
responses and reductions in acute lower respiratory infection and diarrhea to nutrient
supplementation in malnourished or infected young children [25-31]. Thus the limited data available on the relationship between micronutrient intake
and supplementation on immune function in children is consistent with data collected
in adult populations: supplemental micronutrients are efficacious among those suffering
from syndromes related to immune dysfunction but unlikely to augment immune function
or decrease infection risk among well nourished and healthy individuals.

Among our "medium" and "high" supplement groups, ingestion of the fortified breakfast
cereal represented ~20-85% of total dietary intake for zinc, iron, vitamin B6, folate
(and other B complex vitamins), and antioxidant vitamins. Supplemental capsules containing
a micronutrient mixture would have provided a greater quantity, but this study was
designed to test the utility and efficacy of using a fortified breakfast cereal delivery
system. The volume of breakfast cereal consumed per day by our young subjects (mean
of ~60 g or 2.7 cups) over a two month period approached the maximum amount they could
tolerate. For the five key immune-related nutrients in the "high" fortification group--vitamins
A, C, and E, zinc, iron--supplementation with the fortified cereal product by the
children during the study provided approximately 30% of the U.S. Daily Value above
typical food intake. Thus at the nutrient density used in this study, the strategy
of using fortified breakfast cereal to improve immune function and lower URTI risk
in healthy children is not recommended.

Conclusions

In conclusion, our hypothesis of URTI incidence reduction and immune modulation in
healthy, school-aged children through ingestion of a breakfast cereal fortified with
immune-related nutrients was not supported. We measured URTI rates and conducted a
variety of in vitro and in vivo measures of both innate and adaptive immune function, and failed to find any significant
group differences during a 2-month winter period. Thus within the context of this
investigation, our data do not support the use of immune benefit claims for healthy,
school-aged children from ingestion of fortified breakfast cereal.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

NDC was the primary investigator for this study, managed all aspects of subject recruitment
and scheduling, data collection, sample analysis, and manuscript preparation. HDA
(immunologist) coordinated all sample collection and immune assays, and participated
in preparation of the manuscript. SW assisted in the statistical analysis. All authors
have read and approved the final manuscript

Acknowledgements

Supported by a grant from the General Mills Bell Institute of Health and Nutrition,
Minneapolis, MN. The authors have no conflicts of interest.